Yeast organisms produce a number of proteins synthesized intracellularly, but having a function outside the cell. Such extra-cellular proteins are referred to as secreted proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-protein form containing a presequence ensuring effective direction of the expressed product across the membrane of the endoplasmatic reticulum (ER). The presequence, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the protein is transported to the Golgi apparatus. From the Golgi the protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium (Pfeffer, S. R. and Rothman, J. E. Ann. Rev. Biochem. 56 (1987) 829-852).
Several approaches have been suggested for the expression and secretion in yeast of proteins heterologous to yeast. European published patent application No. 0088632A describes a process by which proteins heterologous to yeast are expressed, processed and secreted by transforming a yeast organism with an expression vehicle harbouring DNA encoding the desired protein and a signal peptide, preparing a culture of the transformed organism, growing the culture and recovering the protein from the culture medium. The signal peptide may be the desired proteins own signal peptide, a heterologous signal peptide or a hybrid of native and heterologous signal peptide.
A problem encountered with the use of signal peptides heterologous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage after the signal peptide.
The S. cerevisiae MF.alpha. (.alpha.-factor) is synthesized as a prepro form of 165 amino acids comprising a 19 amino acid long signal- or prepeptide followed by a 64 amino acid long "leader-" or propeptide, encompassing three N-linked glycosylation sites followed by (LysArg(Asp/Glu, Ala).sub.2-3 .alpha.factor).sub.4 (Kurjan, J. and Herskowitz, I. Cell 30 (1982) 933-943). The signal-leader part of the preproMF.alpha. has been widely applied to obtain synthesis and secretion of heterologous proteins in S. cerevisiae.
Use of signal/leader peptides homologous to yeast is known from a. o. U.S. Pat. No. 4,546,082, European published patent applications Nos. 011620A, 0123294A, 0123544A, 0163529A, and 0123289A and DK patent specifications Nos. 2484/84 and 3614/83.
In EP 0123289A utilization of the S. cerevisiae a-factor precursor is described whereas DK 2484/84 describes utilization of the Saccharomyces cerevisiae invertase signal peptide and DK 3614/83 utilization of the Saccharomyces cerevisiae PH05 signal peptide for secretion of foreign proteins.
U.S. Pat. No. 4,546,082, EP 0016201A, 0123294A, 0123544A, and 0163529A describe processes by which the .alpha.-factor signal-leader from Saccharomyces cerevisiae (MF.alpha.1 or MF.alpha.2) is utilized in the secretion process of expressed heterologous proteins in yeast. By fusing a DNA-sequence encoding the S. cerevisiae MF.alpha. signal/leader sequence to the 5'end of the gene for the desired protein secretion and processing of the desired protein was demonstrated.
A number of secreted proteins are routed so as to be exposed to a proteolytic processing system which can cleave the peptide bond at the carboxy end of two consecutive basic amino acids. This enzymatic activity is in S. cerevisiae encoded by the KEX 2 gene (Julius, D. A. et al., Cell 37 (1984b), 1075). Processing of the product by the KEX 2 gene product is needed for the secretion of active S. cerevisiae mating factor .alpha. (MF.alpha. or .alpha.-factor) but is not involved in the secretion of active S. cerevisiae mating factor a.
The growing evidence for secretion being a default route in the localization of proteins in eucaryotes, where conformation of the protein to be secreted or its precursor is the essential parameter for the efficiency of the process (Pfeffer, S. R. and Rothman, J. E., Ann.Rev.Biochem. 56 (1987) 829-852), elicited a search for more efficient leaders than the MF.alpha. leader for secretion in S. cerevisiae.
Accordingly it is an object of the present invention to provide more efficient leaders than the .alpha.-factor leader for the secretion in yeast of small proteins.
In competition experiments of the type described by M. Egel-Mitani et al., GENE (1988) (in press) with insulin precursors of the type described in EP-patent application No. 163,529 we found a limiting factor in expression and secretion of the insulin precursors to be caused by the N-terminal of the precursor, most likely due to inefficient processing at the lysine-arginine sequence separating the signal-leader from insulin precursor.
A further object of the present invention is to provide a secretion system for heterologous proteins in yeast ensuring a highly efficient processing of the mature protein by means of a KEX 2 encoded endopeptidase at a dibasic sequence thus improving the yield of the mature product.